The action of glutamate-activated ion channels determines the flow of [unreadable] information via excitatory synapses throughout the mammalian brain.[unreadable] As a result, the normal function and regulation of glutamate channels[unreadable] (of the AMPA, kainate and NMDA subtypes) are involved in virtually[unreadable] all brain functions. In the past 10-15 years, fundamental studies of N-[unreadable] methyl-D-aspartate (NMDA) receptors provide one of the clearest[unreadable] rationales for the relevance of basic research to clinical problems.[unreadable] These studies have provided new insights into normal brain functions[unreadable] such as synaptic plasticity, the formation of memories, and the action[unreadable] of psychomimetic drugs such as phencyclidine (PCP) on human[unreadable] behavior. Excessive stimulation of glutamate receptors can cause[unreadable] neuronal cell death in seizures and stroke, and may play an important[unreadable] role other neuropsychiatric illnesses. An amazing complexity of[unreadable] regulatory mechanisms influence glutamate receptors. For example,[unreadable] NMDA receptors are regulated by allosteric mechanisms, multiple[unreadable] kinases, phosphatases and soluble second messengers. Although such[unreadable] complexity may seem fitting given the central role of excitatory[unreadable] synapses, the question of what determines the specificity of such[unreadable] interactions is unexplored. Calcium influx into neurons through open[unreadable] NMDA channels at synapses initiates several of these regulatory[unreadable] mechanisms, thus we have focused on the regulation of hippocampal[unreadable] NMDA receptors by intracellular calcium. Our results suggest that[unreadable] compartmentalization and local interactions between glutamate[unreadable] receptors, regulatory proteins and cytoskeletal elements in the[unreadable] postsynaptic density (PSD) are keys to this puzzle. These interactions[unreadable] are likely to affect the activity of synaptic NMDA channels as well as[unreadable] the formation and receptor composition of hippocampal synapses. We[unreadable] will test two aspects of this general hypothesis in this proposal. First,[unreadable] we will examine the domains of the NMDA receptor responsible for[unreadable] calcium regulation (Aim 1-2) and desensitization (Aim 3). Preliminary[unreadable] results demonstrate that calcium regulation is NR2A specific and[unreadable] chimeric/deletion constructs suggest regions of NR1 and NR2A that are[unreadable] involved, perhaps by a ball-and-chain mechanism. We will also[unreadable] examine the possible inductive role of NMDA receptors, and the NR2B[unreadable] subunit in particular, in the function and localization of individual[unreadable] synapses on hippocampal neurons (Aim 4). These studies will make[unreadable] use of transgenic mice lacking the NR2B subunit. The proposed[unreadable] studies will use recombinant NMDA receptors expressed in 293 cells[unreadable] and Xenopus oocytes as well as native receptors in cultured[unreadable] hippocampal neurons. Novel methods we developed for studies of[unreadable] synaptic NMDA receptors and function of individual synaptic sites will[unreadable] be used.